The Optimal Supplementation of Fermented Product Produced by Bacillus subtilis Strain LYS1 with High Surfactin Yield for Improving Growth Performance, Intestinal Villi Morphology, and Tibial Bone Strength in Broilers

Simple Summary Bacillus subtilis is a probiotic that can produce surfactin during fermentation. Surfactin is a lipopeptide whose antibacterial activity increases positively with fatty acid chain length, showing great potential to replace antibiotic growth promoters. B. subtilis LYS1 (LYS1) is selected as a strain that can produce high surfactin yields. In addition, previous research has confirmed that surfactin content and LYS1 counts in the fermented product (FP) are positively correlated with weight gain and production efficiency factor in broilers. This study shows that dietary supplementation with 1 to 2.5% LYS1 FP can improve the growth performance, development of intestinal villi, and tibial bone density/mineralization in broilers, and that 1.9% is the optimal supplementation level of LYS1 FP in the diet. Abstract This study aimed to investigate the physiochemical characterizations of the fermented product (FP) produced by the high-yield surfactin strain Bacillus subtilis LYS1 (LYS1), as well as its effects on growth performance, carcass traits, intestinal morphology, tibial bone characteristics, and clinical blood biochemistry in broilers. Accordingly, the optimal supplementation of FP for improving growth performance, intestinal villi development, and tibial bone strength in broilers was elucidated using a broken-line quadratic (BLQ) ascending model. Three hundred and sixty 0-day-old Ross 308 broiler chicks, with equal numbers of both sexes, were randomly assigned to dietary supplementation of 2.5% fish meal or 0, 1, 1.5, 2, or 2.5% FP. Each treatment had six replicates, and the experimental period was 5 wk. The LYS1 count, surfactin content, and surfactin composition of the FP were 9.1 log CFU/g, 11.23 mg/g, and C12 to C18, respectively. The FP-supplemented groups improved feed intake, weight gain (WG), and production efficiency factor at 0 to 5 weeks old (p < 0.05) compared with the 0% group. The villus height/crypt depth (V/C) in the jejunum and ileum of the FP-supplemented groups was higher than in the 0% group (p < 0.05). The tibiotarsal index, Ca, and P in the tibia showed a linear effect with increased FP supplementation (p < 0.05). Moreover, the tibiotarsus weight/length index (TWLI) showed a quadratic effect with increased FP supplementation (p < 0.05). The optimal supplementation of FP for WG, V/C in the jejunum and ileum, and TWLI was 1.8, 1.9, and 1.6%, respectively. In conclusion, dietary supplementation with 1 to 2.5% LYS1 FP in broilers can improve their growth performance and the development of intestinal villi. Moreover, 1.9% is the optimal supplementation of LYS1 FP in the diet, based on the fitting results obtained with the BLQ model.


Introduction
Bacillus subtilis can generate endospores and has better survival ability than other probiotics during the feed process.Moreover, in terms of the practical functions of B. subtilis in animal guts, it has been reported that their endospores can resist low pH values, bile salts, and other digested enzymes [1,2].B. subtilis is normally applied in the poultry industry as a direct-fed microbial, improving the growth performance, development of intestinal villi, and tibial health in broilers [3,4].Surfactin, a secondary metabolite produced by Bacillus spp.through the non-ribosomal peptide synthetase, is an amphiphilic lipopeptide.In general, surfactin consists of fatty acid chains ranging in length from 12 to 17 carbon atoms, where C 14 and C 15 account for the highest proportions in its composition [5].The antibacterial activity of surfactin increases positively with fatty acid chain length [6].Accordingly, the diverse combinations of surfactin have a wide range of antibacterial activities, showing great potential to replace antibiotic growth promoters [7][8][9].
Recent studies have indicated that directly feeding poultry with Bacillus spp.fermented product (FP) or powder produced by their strains, which contains surfactin, can improve the development of intestinal villi, adjust the intestinal microflora, and promote the growth of broilers [10][11][12].Cheng et al. [11] reported that supplementation with 0.3% B. subtilis NIU068 FP (surfactin content: 0.3 mg/kg feed) could improve growth performance, intestinal villus height, and crypt depth in broilers.Chen and Yu [13] found that supplementation with 0.3% B. licheniformis FP (surfactin content: 14.1 mg/kg feed) could improve the ADG at 1 to 35 d old and adjusted the abundance of the genus Lactobacillus in feces, which was positively correlated with ADG.Nevertheless, few studies have investigated the effects of surfactin content and its composition in FP on growth and physiology in broilers simultaneously.
B. subtilis LYS1 (LYS1) is a selected strain known for its ability to produce high yields of surfactin, as previously reported by the authors [4].The surfactin content in FP produced by LYS1 through solid-state fermentation for 2 days exceeded 10 mg/g and included surfactin compositions from C 12 to C 17 , with a notable 6.96% proportion of long carbon chains (C 16 and C 17 ), which was higher compared to other FPs produced by selected Bacillus strains.Furthermore, Lee et al. [4] confirmed that surfactin content and Bacillus-like counts in FP were positively correlated with weight gain and production efficiency factor.Consequently, supplementation with 2.5% LYS1 FP improved the growth performance and intestinal villi development in broilers.The above results imply that the surfactin composition and content in the FP have important effects on the growth performance of broilers; thus, analyzing the surfactin characteristics of FP is one of the objectives of this study.The growth performance of broilers is inseparable from their intestinal absorption capacity and bone health.Good intestinal villus physiology can help in effectively utilizing nutrients for body growth, while strong tibial bones can prevent leg diseases to support the rapid growth of broilers [3,14].Guo et al. [15] indicated that supplementation with 0.5% B. subtilis PB6 could improve the overall average daily gain and tibial characteristics.However, the optimal supplementation of LYS1 FP for improving growth performance, intestinal villi morphology, and tibial bone strength in broilers still needs to be clarified.
This study aimed to investigate the physiochemical characterizations and surfactin characteristics of FP with high surfactin content produced by LYS1, along with its effects on growth performance, carcass traits, intestinal morphology, tibial bone characteristics, and clinical blood biochemistry in broilers.Finally, the optimal supplementation of surfactin content and LYS1 counts in FP on improving growth performance, intestinal villi development, and tibial bone strength in broilers was elucidated using a broken-line quadratic (BLQ) ascending model.

FP Preparation
The high-yield surfactin strain LYS1, as identified by Lee et al. [4], was used as the fermented strain for FP preparation.Soybean meal was sterilized at 121 • C at 1.21 kg/cm 2 for 30 min, then cooled down to 45 • C. The LYS1 counts at a concentration of 10 6 CFU/g of substrate were premixed and inoculated.Subsequently, the water content of the substrate was adjusted to 55% in order to facilitate aerobic fermentation at 37 • C for two days.The FP was dried in an oven at 55 • C until the water content was below 12% and was then ground to produce FP powder.

Animal Management and Experimental Design
Three hundred and sixty 0-day-old Ross 308 broiler chicks with initial body weight (BW) of 43 ± 1 g and equal numbers of both sexes, under a randomized complete block design, were randomly assigned to dietary supplementation with 2.5% fish meal or 0, 1, 1.5, 2, or 2.5% FP.Each treatment had six replicates (with 10 broiler chicks per pen).The experimental period was 5 wk.Feed and water were provided ad libitum throughout the experimental period.Diet formulation referred to the recommendation of Ross broilers [16], and the management of the broilers followed the Ross Broiler Management Manual [17].The feed composition analysis is presented in Table 1.The Institutional Animal Care and Use Committee of National Chiayi University (IACUC, protocol number 110018) approved all of the procedures used in this experiment.

The Physiochemical Characterizations of FP
The FP underwent serial dilution in 0.9% NaCl solution and was then placed on tryptic soy agar (HIMEDIA ® , Mumbai, MH, India) at 37 • C for 24 h.The counts of the LYS1 colonies were quantified as colony-forming units per gram (CFU/g).The pH of the FP sample was determined using a pH meter (PB-10, Sartorius ® , Göttingen, Germany), while the water content was assessed with an infrared moisture meter (ML 50, A&D Company, Limited, Toshima, Japan).

The Protease Activity of FP
The protease activity of FP was determined using the methods of Secades and Guijarro [18] and Oguntoyinbo et al. [19].In brief, the crude enzyme solution obtained from the reaction sample was subjected to centrifugation at 15,000× g for 10 min at 4 • C. Subsequently, 100 µL of the supernatant was combined with 100 µL of 0.72 N NaOH (Macron Fine Chemicals™, Center Valley, PA, USA), and the absorbance at a wavelength of 420 nm was recorded to determine the enzyme activity unit (U/g).This unit was defined as the change in absorbance value over time following the interaction of the sample with the substrate.

The Surfactin Analysis of FP
The samples were mixed with methanol (1:10, w/v), and the mixture was centrifuged at 10,000× g for 10 min at 4 • C to remove insoluble matter.Subsequently, the supernatant was filtered with a 0.22 µm polyvinylidene fluoride membrane to obtain a filtrate for surfactin analysis.The qualification and quantification of surfactin in the FP followed the procedure outlined by Lee et al. [4].Analysis was conducted using liquid chromatography electrospray ionization high-resolution mass spectrometry (LC-ESI-HRMS, Thermo Fisher Scientific, Bremen, Germany).Full-scan LC-ESI-HRMS in positive ion mode showed a massto-charge ratio (m/z) ranging from 988 to 1200.The sample was injected at a volume of 5 µL into a reversed-phase column (Mightysil RP-18 GP, 250 × 4.6 mm; 5 µm, Kanto Kagaku, Okayama, Japan) for separation at a flow rate of 1 mL/min over a duration of 0 to 25 min.The mobile phase comprised 90% acetonitrile (Anaqua™ Chemicals Supply, Wilmington, DE, USA) and 10% water (v/v) with 0.1% formic acid (Merck, Hessen, Germany).The m/z of the surfactin standard (Sigma-Aldrich ® , St. Louis, MO, USA) served as the basis for qualitative analysis.Meanwhile, a concentration gradient solution of the surfactin standard was prepared and analyzed under the aforementioned chromatographic conditions in order to establish a standard curve.Subsequently, the surfactin concentration in the FP was determined based on this standard curve.

Feed Composition Analysis
Proximate feed analysis was conducted according to the procedure of AOAC [20] to analyze the moisture (method 930.15), crude protein (method 984.13), ether extract (method 920.39), ash (method 942.05), calcium (method 968.08), and phosphorus (method 965.17) contents.The LYS1 count, protease activity, and surfactin content in the feed were analyzed as described above.

Sample Collection
Twenty-four broilers formed each group, with equal numbers of both sexes, and were euthanized for sample collection following a withdrawal period from feed for 12 h at 5 weeks old.Carcass traits, intestinal morphology, tibial bone characteristics, and clinical blood biochemistry were analyzed in all groups.Each analysis was performed in six replicates (n = 6), with four broilers included in each replicate.

Carcass Traits
Various organs and tissues, including the heart, liver, gallbladder, proventriculus, gizzard, spleen, intestine (from the duodenum to rectum), abdominal fat (from the gizzard to celiac fat), breast (with bone and skin removed), and thigh (from the femur to the tibia, with bone and skin removed), were assessed for weight, and their relative weight in relation to body weight was determined.

Intestinal Morphology
Three-centimeter sections of jejunum located before Meckel's diverticulum and ileum situated after Meckel's diverticulum were procured and preserved in 10% buffered formaldehyde for 48 h.Subsequently, each segment was embedded in paraffin, and a 5 µm slice of each specimen was obtained using a microtome, mounted on a glass slide, and stained with hematoxylin and eosin.The histological sections of the intestinal tissue were analyzed utilizing an optical microscope (Labophot-2, Nikon, Tokyo, Japan).Morphometric parameters were assessed using the ImageJ software (version 1.52v) (http://rsb.info.nih.gov/ij/,accessed on 4 May 2020).Measurements of the villus height, villus width, crypt depth, and tunica muscularis thickness of the small intestine were conducted at 10 different points for each parameter, and the villus height/crypt depth (V/C) ratio was computed.Villus height was determined from the apex of the crypt to the lamina propria of the villus.Villus width was measured from one side to the other at the midpoint of the villus.Crypt depth was defined as the shortest vertical distance from the point of contact with the villus to the mucous membrane.The thickness of the tunica muscularis was calculated as the distance between the lamina muscularis mucosae internally and the tunica serosa externally.The measurement of villus surface area was performed using the method of Liu et al. [21].In brief, the surface area of the villi = π•w•h (where w represents villus width and h represents villus height).

Tibial Bone Characteristics
The measurement of tibial bone characteristics was performed using the method of Mutuş et al. [22].The labeled drumsticks were boiled at 100 • C for 10 min, then cooled to room temperature and manually de-fleshed, with removal of soft tissues and the patella.Then, the tibiotarsal length and bone weight were determined after air-drying for 24 h at room temperature.Before breaking, each bone was marked at its midpoint, and external diameters were measured perpendicular and parallel to the direction of applied force using a caliper.After breaking, diameter measurements were taken inside and outside the mid-shaft of the bone, perpendicular and parallel to the direction of applied force, in order to calculate the area moment of inertia.This parameter was used with elastic deformation to calculate the stress (kg/cm 2 ).The thickness of the medial and lateral walls was measured at the midpoint mark using a dial caliper.The diameter of the medullary canal was determined by subtracting the thicknesses of the medial and lateral walls from the diameter at the diaphysis.The tibiotarsus weight/length, tibiotarsal, and robusticity indexes were calculated by the following formulae: (a) tibiotarsus weight/length index = tibia weight/tibial length; (b) tibiotarsal index = diaphysis diameter − medullary canal diameter/diaphysis diameter × 100; (c) robusticity index = bone length/cube root of bone weight.To analyze ash, Ca, and P contents in the tibia, bones were oven-dried at 105 • C for 24 h and were ashed in a muffle furnace at 600 • C for 6 h.The contents of ash (method 942.05),Ca (method 968.08), and P (method 965.17) in the tibia were analyzed as described by AOAC [20].

Clinical Blood Biochemistry
Blood samples were taken from the brachial vein of broilers, followed by the collection of serum after centrifugation of the blood samples at 1734× g for 15 min.The serum was then preserved at −20 • C for subsequent analysis.The blood biochemistry of the serum was assessed, encompassing the activities of enzymes, such as aspartate aminotransferase (AST), lactate dehydrogenase (LDH), creatine kinase (CK), alkaline phosphatase (ALKP), and the levels of various components, including total protein (TP), albumin (ALB), blood urea nitrogen (BUN), uric acid (UA), total cholesterol (TC), triacylglyceride (TG), calcium (Ca), and phosphate (P).This analysis was conducted using an automated blood chemical analyzer equipped with Roche testing kits (Roche Cobas Mira Plus, Switzerland).Additionally, the albumin-to-globulin (A/G) ratio was calculated.The enzymatic activities and concentrations of the analyzed parameters were determined spectrophotometrically according to the methodology outlined by Akiba et al. [23].

Statistical Analysis
The continuous variables in the physiochemical characterizations, protease activity and surfactin content of FP, growth performance, carcass traits, intestinal morphology, tibial bone characteristics, and clinical blood biochemistry were analyzed according to the following statistical model: where Y ij represents the measured value on the i-th treatment in the j-th block, µ is the overall mean, τ i is the fixed effect of the i-th treatment, β j is the random effect of the j-th block, and e ij is the random error associated with Y ij .The data were analyzed using the MIXED procedure (SAS 9.4), the groups were compared using a one-way ANOVA with a Tukey post hoc test, and coefficients for linear and quadratic contrasts were determined using the IML procedure (SAS 9.4) for the effect of LYS1 FP supplementation, with p < 0.05 indicating a statistically significant difference.The survival rate of growth performance was analyzed using the NPAR1WAY procedure (SAS 9.4).The groups were compared using SAS ® macro implementation of a multiple comparisons test as described by Elliott and Hynan [24], with p < 0.05 indicating a statistically significant difference.The BLQ ascending model was fitted to growth performance, intestinal villi morphology, and tibial bone characteristics to further estimate the optimal LYS1 FP supplementation, and the results were analyzed according to the following statistical model: where Y ij represents the observed value associated with the pen randomly assigned to the FP supplementation i within block j, φ is the unknown maximum response (plateau) under the BLQ model, β 1 and β 2 are the corresponding unknown regression coefficients describing the relationship between X i and Y ij for values of X i smaller than the plateau, ω is the unknown minimum level of FP supplementation to reach the plateau under the BLQ model, X i is the i-th known FP supplementation, β j is the random effect of the j-th block, and e ij is the random error associated with the experimental unit in the j-th block that received the i-th FP supplementation.The data were analyzed using the NLMIXED procedure (SAS 9.4).

The Physiochemical Characterizations and Functional Components of LYS1 FP
Table 2 shows the physiochemical characterizations of LYS1 FP.The LYS1 count and pH value increased to 9.36 log CFU/g and pH 7.35 (p < 0.05), respectively, after one or two days of fermentation, and then decreased (p < 0.05) after the drying and grinding treatment.The LYS1 count, protease activity, and surfactin content of LYS1 FP were 9.10 log CFU/g, 305 U/g, and 11.23 mg/g, respectively.

Growth Performance
Table 4 shows the effects of LYS1 FP supplementation on growth performance in broilers.At 0 to 3 wk, the FI of the 1.5% FP, 2.5% FP, and fish meal groups was higher than that of the 0% group (p < 0.05), and the WG of the 2% FP, 2.5% FP, and fish meal groups was higher than that of the 0% group (p < 0.05).In addition, only the 2% FP group showed an improved PEF (p < 0.05) compared with the 0% group.At 3 to 5 wk, the FI of the FP-supplemented and fish meal groups was higher than that of the 0% group (p < 0.05), and only the FP-supplemented groups showed an improved WG (p < 0.05) compared with the 0% group.At 0 to 5 wk, the FI and WG of the FP-supplemented and fish meal groups were higher than those of the 0% group (p < 0.05), and the FP-supplemented groups showed an improved PEF (p < 0.05) compared with the 0% group.The FI, WG, and PEF at 0 to 3, 3 to 5, and 0 to 5 weeks old showed a linear effect with increased FP supplementation (p < 0.05), and the FI and WG at 3 to 5 weeks old, BW at 5 weeks old, and WG at 0 to 5 weeks old showed a quadratic effect with increased FP supplementation (p < 0.05).

Carcass Traits
Table S1 shows the effects of LYS1 FP supplementation on carcass traits in broilers.The relative weight of the carcass, heart, liver and gallbladder, gizzard and proventriculus, spleen, intestine, abdominal fat, skinless breast, and whole legs showed no significant differences between groups (p > 0.05).

Intestinal Morphology
Table 5 shows the effects of LYS1 FP supplementation on intestinal morphology in broilers.In the jejunum, the villus height and V/C of the FP-supplemented and fish meal groups were higher than those of the 0% group (p < 0.05).In addition, the 2% FP group showed an improved surface area of villus (p < 0.05) compared with the 0% group.The villus height, crypt depth, villus width, V/C, and surface area of villus in the jejunum showed a linear effect with increased FP supplementation (p < 0.05), and the villus height and V/C showed a quadratic effect with increased FP supplementation (p < 0.05).In the ileum, the V/C of the FP-supplemented and fish meal groups was higher than that of the 0% group (p < 0.05), and only the 2% FP group showed an improved V/C (p < 0.05) compared with the fish meal group.In addition, the FP-supplemented and fish meal groups showed an improved crypt depth (p < 0.05) compared with the 0% group.The villus height, crypt depth, V/C, and surface area of villus in the ileum showed a linear effect with increased FP supplementation (p < 0.05), and the crypt depth and V/C showed a quadratic effect with increased FP supplementation (p < 0.05).

Morphometric Parameters, Bone Strength Measurements, and Mineral Contents of the Tibia
Table 6 shows the effects of LYS1 FP supplementation on the morphometric parameters, bone strength measurements, and mineral contents of the tibia in broilers.The force of the 2% FP, 2.5% FP, and fish meal groups was higher than that of the 0% group (p < 0.05).In addition, only the 2.5% FP group showed an improved tibiotarsal index (p < 0.05) compared with the 0% group.The force, tibiotarsal index, Ca, and P in the tibia showed a linear effect with increased FP supplementation (p < 0.05).Moreover, the tibial weight and tibiotarsus weight/length index showed a quadratic effect with increased FP supplementation (p < 0.05).

Clinical Blood Biochemistry
Table 7 shows the effects of LYS1 FP supplementation on clinical blood biochemistry in broilers.The AST and LDH activities of the 2% FP group were higher than those of the 0% group (p < 0.05), and the CK activity of the 0% group was higher than that of the 1.5-2.5% FP and fish meal groups (p < 0.05).The A/G of the 2.5% FP group was higher than that of the 2% FP group (p < 0.05).The activities of AST, LDH, and CK, as well as the contents of TC and TG showed a linear effect with increased FP supplementation (p < 0.05).

The optimal Supplementation of LYS1 FP for Growth Performance, Intestinal Morphology, and Tibial Bone Characteristics
Table 8 shows the optimal supplementation of LYS1 FP for growth performance, intestinal morphology, and tibial bone characteristics in broilers.The fitted broken-line quadratic models of the optimal supplementation of LYS1 FP for growth performance, intestinal morphology, and tibial bone characteristics in broilers are displayed in Table S2.
In terms of growth performance, the optimal supplementation of LYS1 FP for BW at 5 weeks old and WG at 3-5 and 0-5 weeks old was 1.8%, and the optimal supplementation for FI at 3-5 weeks old was 0.9%.In terms of intestinal morphology, the optimal supplementation of LYS1 FP for villus height in the jejunum and crypt depth in the ileum was 1.8 and 1.7%, respectively.The optimal supplementation for V/C in the jejunum and ileum was 1.9%.In terms of tibial bone characteristics, the optimal supplementation of LYS1 FP for tibial weight was 1.5%, and the optimal supplementation for tibiotarsus weight/length index was 1.6%.

Discussion
The variations in Bacillus-like counts and pH values are often used as indicators to evaluate fermentation quality.The counts of the Bacillus strain increased during fermentation, indicating that the strain was suitable for growth under fermentation conditions.The water content of the substrate decreased during the drying and grinding processes, which resulted in decreases in overall water activity and Bacillus-like counts.Nevertheless, Bacillus endospores can resist high temperatures and dry conditions during processing [2,25].The pH value increases during fermentation because the Bacillus strain degrades the protein of the substrate to produce amines and ammonia, and the high temperature of the drying and grinding processes causes the ammonia to evaporate with the water, reducing the pH of the FP [4,14].The surfactin yield of the FP is positively related to the density of the production strain [26].In a previous study, LYS1 was selected as a strain that could produce high surfactin yields.However, the water content of the substrate dropped to 33% when fermented for 2 days, reducing the water activity and affecting the fermentation results of LYS1 [4].In this study, the improved ventilation equipment used in the fermentation process reduced the water evaporation so that the water content of the substrate was maintained at 50-55% during fermentation.Therefore, the LYS1 counts increased by 608% (9.1 vs. 8.25 log CFU/g FP), which indirectly increased the surfactin content of the FP by 5.05% (11.23 vs. 10.69 mg/g FP) compared with the previous study.
The type and proportion of adducts affect the quantitative and qualitative results of surfactins [27,28].This study used the positive ion mode in LC-ESI-HRMS to improve the analysis method and was able to detect the C 12 to C 18 surfactin isoforms in LYS1 FP.Moreover, the sodium and potassium adducts were detected in LYS1 FP, except for the proton adducts.The composition types and ratios of surfactin produced by B. subtilis differ according to the strain characteristics and fermented conditions, and the bioactivity of surfactin increases with the extension of the β-hydroxy fatty acid chains [29][30][31].Dhanarajan et al. [6] indicated that the minimum inhibitory concentration of C 16 surfactin for Escherichia coli was 3.75 µg/mL, which was much lower than that of C 13 surfactin (>120 µg/mL).Compared with the study of Lee et al. [4], this study found reduced water evaporation during fermentation.Consequently, this increased the content of LYS1 surfactin and affected the surfactin composition, and a new long-carbon-chain C 18 surfactin was found.
Supplementation with 1 to 2.5% LYS1 FP showed better WG, FI, and PEF at 0 to 5 weeks old (p < 0.05), which was similar to the results of Lee et al. [4]; supplementation with 2.5% LYS1 FP could replace the same proportion of fish meal in the broilers' diet, and the WG and PEF of the LYS1 group were better than those of the unfermented group.Thus, this study again confirmed the improved effects of LYS1 FP on the growth performance of broilers, which was effectively improved by just supplementation with 1% LYS1 FP, and the improvement showed a linear effect with increased FP supplementation (p < 0.05).In addition, the FI and WG at 3 to 5 weeks old and the WG at 0 to 5 weeks old showed a quadratic effect with increased FP supplementation (p < 0.05), indicating that an optimal supplementation with LYS1 FP for FI and WG in broilers exists.B. subtilis is generally used as a direct-fed microbial applied in diets to improve growth and intestinal health in poultry production [2,32,33].Feed substrates fermented by B. subtilis can transform macromolecular nutrients into more digestible micromolecular ones and reduce the antinutritional factors to improve the utilization of nutrients by broilers [34,35].B. subtilis can produce functional components such as extracellular enzymes (e.g., protease) and antibacterial peptides (e.g., surfactin) during fermentation, which can improve the intestinal health of broiler chickens during their early growth period [2,36].The authors' previous study confirmed that the Bacillus-like counts, protease activity, and surfactin content of FP were moderately correlated with the growth performance of broilers [4].The newly discovered long-carbonchain C 18 surfactin can increase the antibacterial activity of LYS1 FP in broilers' intestines.Therefore, B. subtilis FP not only improves the utilization of substrate nutrients but also provides the benefits of probiotics and secondary metabolites, which promote broilers' intestinal morphology and growth performance [12,14].
B. subtilis can produce particular flavors according to different fermented conditions, influencing the palatability of the FP to poultry [37][38][39].Fish meal contains rich amino acid compositions and easily digestible proteins, is often used for high-quality protein in poultry diets, and affects palatability according to its quality [14,40].This study used the high-yield surfactin strain LYS1 to produce the FP, and the fish meal was a high-quality brand (TripleNine Group) with CP 70%.The FP-supplemented and fish meal groups showed an increased FI at different growth periods (p < 0.05), showing that the LYS1 FP and fish meal had better palatability to the broilers, thereby improving their WG.A higher PEF value means better economic profits in poultry production [41,42].The content and composition of surfactin and the LYS1 counts of LYS1 FP were better than the results of Lee et al. [4].At 0 to 5 wk, the WG of the FP-supplemented groups was better than that of the 0% group (p < 0.05), which could achieve the same performance as the fish meal group.Consequently, supplementation with 1 to 2.5% LYS1 FP improved the PEF at 0 to 5 weeks old (p < 0.05), compared with the 0% group.This shows that LYS1 FP has excellent potential for use in the production of broilers throughout their entire growth period.
The authors' previous study indicated that supplementation with 2.5% LYS1 FP or fish meal did not affect the relative weight of the carcass, organs, and tissues in broilers [4].Lee et al. [14] indicated that supplementation with 5% FP produced by B. amyloliquefaciens CU33 did not affect the relative weight of the carcass, heart, liver, abdominal fat, skinless breast, and whole legs in broilers.These findings are similar to the results of this study, which showed that supplementation with 1 to 2.5% LYS1 FP did not show any negative effects on broilers' carcass traits.
The V/C is generally an indicator of intestinal digestibility [43,44].A higher villus height means a larger intestinal surface area and mature epithelial cells, improving nutrient absorption.Furthermore, a shallower crypt depth means reduced epithelial cell proliferation and reduced energy consumption.The surface area of villus is the curved area of villus height multiplied by villus width and π; a higher value means a more extensive contact surface area between villi and nutrients.Thus, the higher the V/C and surface area of villus, the better the digestibility and absorption of nutrients in the small intestine.Most studies indicate that supplementation with B. subtilis or its FP can improve the villus height, crypt depth, villus width, V/C, and surface area of villus in the small intestine [11,45,46].The authors' previous study found that supplementation with 2.5% LYS1 FP improved villus height, crypt depth, and V/C in the jejunum compared with an unfermented group [4].In this study, supplementation with 1 to 2.5% LYS1 FP improved the villus height, crypt depth, V/C, and surface area of villus (p < 0.05) compared with the 0% group.This again confirms the improved effects of LYS1 FP on intestinal villus development, thereby improving the growth performance of broilers (Table 4).
The digestion and absorption of starch, protein, and fat in broilers mainly occur in the jejunum, while the digestion and absorption of some residual nutrients may occur in the ileum, but the primary function of this intestinal segment is to absorb water and minerals [47,48].Supplementation with 1 to 2.5% LYS1 FP or 2.5% fish meal improved the V/C in the jejunum and ileum (p < 0.05), and supplementation with LYS1 FP could improve the surface area of villus in the jejunum (p < 0.05).The villus height, crypt depth, V/C, and surface area of villus in the jejunum and ileum showed a linear effect with increased FP supplementation (p < 0.05), and the villus height in the jejunum, crypt depth in the ileum, and V/C in both the jejunum and ileum showed a quadratic effect with increased FP supplementation (p < 0.05).This indicates that an optimal supplementation of LYS1 FP exists for villus in the jejunum, crypt depth in the ileum, and V/C in the jejunum and ileum in broilers.
The higher the tibiotarsus weight/length index, the higher the density of the tibia.A higher tibiotarsal index represents better mineralization of the tibia [3,21].In this study, supplementation with 1 to 2.5% LYS1 FP could linearly improve the force and tibiotarsal index in the tibia (p < 0.05), compared with the 0% group.Moreover, the tibiotarsus weight/length index showed a quadratic effect with increased FP supplementation (p < 0.05).These results indicate that LYS1 FP improved the mineralization and bone strength in the tibia, helping to support the heavier BW of broilers (Table 4).Ca and P are essential elements that constitute hydroxyapatite, the main component of bone, and are related to compressive strength [49].Most studies indicate that adding B. subtilis to broilers' diets can improve the strength, length, weight, index, and mineral composition (Ca and P) of the tibia [3,15,50].Similarly, the FP-supplemented groups showed a linear increase in Ca and P contents in the tibia (p < 0.05), showing that LYS1 FP improved the accumulation capacity of Ca and P in bones.Supplementation with B. subtilis improved the mineralization and bone strength in the tibia, related to the improvements in the intestinal digestion of nutrients and absorption of Ca and P [3,51].This study found that supplementation with LYS1 FP could improve the V/C in the ileum (p < 0.05), increasing the absorption of Ca and P in the ileum to improve tibial mineralization.Surfactin is an antibacterial lipopeptide produced by B. subtilis; its heptapeptide ring contains two acidic amino acids (Glu 1 and Asp 5 ), forming a "claw"-like structure.The pKa of these two amino acid residues is 5.4, above which they will dissociate; therefore, this structure is a potential binding site for divalent cations [52].In the alkaline environment of the small intestine, in addition to its original antibacterial function, surfactin may also help broilers to absorb Ca and other minerals.This hypothesis needs further exploration to confirm it.This study found that adding FP produced by the high-yield surfactin strain LYS1 to broilers' diet could improve the mineralization, density, stiffness, and composition (Ca and P) of the tibia, and the improvement showed a linear effect with increased FP supplementation (p < 0.05).This means LYS1 FP can improve the development of the tibia to support a faster increase in BW.The tibial weight and tibiotarsus weight/length index showed a quadratic effect with increased FP supplementation (p < 0.05), meaning that an optimal supplementation of LYS1 FP exists for improving tibial weight and tibiotarsus weight/length index in broilers.
Clinical blood biochemistry often uses blood enzymes to detect liver and muscle damage in animals.AST and LDH are widely distributed in broilers' various tissues, and injury to the liver and skeletal muscle will increase AST and LDH activity.In addition, CK is a specific indicator of muscle cell damage [53].Among these enzymes, the half-life order is AST > CK > LDH.When LDH activity increases, but CK activity does not increase in serum, there may be no muscle damage [54].However, it should be noted that blood enzyme profiles can only be used as a rough indicator to explain elevated blood enzyme activity, and they do not represent the condition of a specific organ [55].The authors' previous study indicated that supplementation with 2.5% LYS1 FP did not affect the enzyme activity in clinical blood biochemistry in broilers [4]; this is not consistent with the results of this study.When the enzymes enter blood circulation, due to the different removal rates of various components, the composition of the enzyme profile may be changed and is affected by feed ingredients, nutritional status, season, gender, age, feeding, and management.The activities of AST and CK increase in commercial broilers due to the rapid growth rate of skeletal muscle mass [56].Generally, when muscles are injured, CK activity increases first, followed by AST activity.When CK activity decreases and AST activity increases, it may indicate a muscle healing process.In addition, if muscle cells are dystrophic, their CK activity may also be increased [57].LDH activity can be used to evaluate muscle metabolism, and AST activity may be affected by the body's protein metabolism rate [58].In this study, supplementation with 2% LYS1 FP showed higher WG (Table 4), meaning that the broilers were in a rapid growth period.Therefore, the 2% FP group showed higher activity of AST and LDH than the 0% group (p < 0.05), while the CK activity of the 1.5-2.5% FP and fish meal groups was lower than that of the 0% group (p < 0.05).This may have been because the muscles were already in the repair process.In addition, the FI of the 0% group was lower than that of the other groups (p < 0.05), which may have affected the physiological nutritional status of their muscles.
The contents of TP, ALB, TC, and TG in the blood may be affected by the FI and the diet composition.Yaman et al. [59] indicated that the contents of TP and ALB in broilers were reduced after restricted feeding.In this study, the FI of the 0% group was lower than that of the other groups (p < 0.05); thus, the 0% group showed lower contents of TP and ALB.The A/G of the 2.5% FP group was higher than that of the 2.0% FP group (p < 0.05), but there was no difference between them and the 0% group (p > 0.05), indicating that the blood protein composition in each group was in a normal state.The UA is the primary end product of nitrogen metabolism in poultry, which is often used together with BUN to evaluate the renal function of poultry [55].Rajman et al. [60] indicated that broilers had lower contents of TC and TG after restricted feeding, similar to the results of the present study.The activity of AST and LDH in broilers, along with their contents of TC and TG, increased linearly with increased FP supplementation (p < 0.05), while the activity of CK decreased linearly (p < 0.05).This is consistent with the BW and FI in broilers increasing linearly with increased FP supplementation (p < 0.05) (Table 4).In addition, surfactin is a biosurfactant with the property of emulsifying oil [61].LYS1 FP had a higher surfactin content, which may be related to its linear effect on increasing the levels of TC and TG in serum (p < 0.05), but further exploration is needed in order to confirm this.Recent studies have found that feeding broilers with FP containing surfactin can improve their growth performance.Cheng et al. [11] found that supplementation with 0.3% B. subtilis NIU068 FP (the endospore count was 3.6 × 10 6 CFU/kg feed, and the surfactin content was approximately 0.3 mg/kg feed) could improve BW and intestinal villus morphology in broilers, compared with the control group.Chen and Yu [13] found that supplementation with 0.3% B. licheniformis FP (the B. licheniformis count was 9 × 10 12 CFU/kg feed, and the surfactin content was approximately 14.1 mg/kg feed) could improve broilers' WG and regulated their fecal microbial flora.The authors' previous study indicated that supplementation with 2.5% LYS1 FP (the LYS1 count was 9.64 log CFU/kg feed, and the surfactin content was 276 mg/kg feed) could improve growth performance in broilers, and the surfactin content and Bacillus-like counts of the FP were moderately correlated with growth performance [4].
This study found that adding 1 to 2.5% LYS1 FP (the LYS1 count was 10.10-10.49log CFU/kg feed, and the surfactin content was 112.30-280.75 mg/kg feed, Table 1) to broilers' diets could improve WG, V/C in the jejunum and ileum, and tibial mineralization.The optimal supplementation of LYS1 FP for WG is 1.8%, consistent with the finding that adding more than 2% LYS1 FP leads to heavier WG.The optimal supplementation of LYS1 FP for FI is 0.9%, implying that adding more than 1% LYS1 FP can increase FI in broilers.The optimal supplementation of LYS1 FP for V/C in the jejunum and ileum is 1.9%.This is consistent with the finding that adding more than 2% LYS1 FP leads to a better V/C, which can reduce intestinal energy consumption and increase the absorption of nutrients, thereby improving growth performance, and 2% LYS1 FP is optimal.The optimal supplementation of LYS1 FP for the tibiotarsus weight/length index is 1.6%, implying that adding more than 2% LYS1 FP leads to better tibial density, thereby supporting rapid weight growth in broilers.Based on the above fitting results obtained by the BLQ model, the optimal supplementation of LYS1 FP for broilers' diets was adjusted to 1.9% (LYS1 count of 10.37 log CFU/kg feed, and surfactin content of 213.37 mg/kg feed), which can simultaneously improve the WG, V/C in the jejunum and ileum, and tibial bone density in broilers.

Conclusions
The LYS1 counts, surfactin content, and surfactin composition of the FP were 9.1 log CFU/g, 11.23 mg/g, and C 12 to C 18 , respectively.Supplementing 1 to 2.5% LYS1 FP to the diet of broilers can improve their growth performance, the development of intestinal villi, and tibial bone density/mineralization.Based on the fitting results obtained with the BLQ model, the optimal supplementation of LYS1 FP is 1.9% for WG, intestinal villi morphology, and tibia development in broilers.

Supplementary Materials:
The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/ani14142079/s1,Table S1: Effect of LYS1 FP supplementation on carcass traits in broilers.;Table S2: The optimal supplementation for LYS1 FP for growth performance, intestinal morphology, and tibial bone characteristics in broilers as estimated based on fitted brokenline quadratic models.Informed Consent Statement: Not applicable.

Table 1 .
Ingredient composition of the basal diets.
a-c Means in the same column with different superscripts are significantly different (p < 0.05).1Dataare the means ± SD of 3 batches of each fermented product (FP), n = 3.2The water content ranged from 55 to 50% during fermentation and was below 12% during the drying and grinding processes.3LYS1FP: fermented product produced by Bacillus subtilis LYS1.

Table 4 .
Effects of LYS1 FP supplementation on growth performance in broilers 1 .
Abbreviations: BW, body weight; FCR, feed conversion ratio; FI, feed intake; PEF, production efficiency factor; SR, survival rate; WG, weight gain.a,bMeans in the same row with different superscripts are significantly different (p < 0.05).1Dataare the means of 6 pens of broilers, each pen with 10 broilers (n = 6). 2 LYS1 FP: fermented product produced by Bacillus subtilis LYS1.3SR in each group was 100%.

Table 5 .
Effects of LYS1 FP supplementation on intestinal morphology in broilers 1 .
a-c Means in the same row with different superscripts are significantly different (p < 0.05).1Dataare the means of 6 pens of broilers (n = 6). 2 LYS1 FP: fermented product produced by Bacillus subtilis LYS1.

Table 6 .
Effects of LYS1 FP supplementation on morphometric parameters, bone strength measurements, and mineral contents of the tibia in broilers 1 .
a, b Means in the same row with different superscripts are significantly different (p < 0.05).1Dataare the means of 6 pens of broilers (n = 6). 2 LYS1 FP: fermented product produced by Bacillus subtilis LYS1.

Table 7 .
Effects of LYS1 FP supplementation on clinical blood biochemistry in broilers 1 .

Table 8 .
The optimal supplementation of LYS1 FP for growth performance, intestinal morphology, and tibial bone characteristics in broilers 1 .